Role of the cytokine-induced SH2 domain-containing protein CIS in growth hormone receptor internalization

The cytokine-inducible SH2 domain-containing protein CIS inhibits signaling from the growth hormone (GH) receptor (GHR) to STAT5b by a proteasome-dependent mechanism. Here, we used the GH-responsive rat liver cell line CWSV-1 to investigate the role of CIS and the proteasome in GH-induced GHR internalization. Cell-surface GHR localization and internalization were monitored in GH-stimulated cells by confocal immunofluorescence microscopy using an antibody directed against the GHR extracellular domain. In GH na?ve cells, GHR was detected in small, randomly distributed granules on the cell surface and in the cytoplasm, with accumulation in the perinuclear area. GH treatment induced a rapid (within 5 min) internalization of GH.GHR complexes, which coincided with the onset of GHR tyrosine phosphorylation and the appearance in the cytosol of distinct granular structures containing internalized GH. GHR signaling to STAT5b continued for approximately 30-40 min, however, indicating that GHR signaling and deactivation of the GH.GHR complex both proceed from an intracellular compartment. The internalization of GH and GHR was inhibited by CIS-R107K, a dominant-negative SH2 domain mutant of CIS, and by the proteasome inhibitors MG132 and epoxomicin, which prolong GHR signaling to STAT5b. GH pulse-chase studies established that the internalized GH.GHR complexes did not recycle back to the cell surface in significant amounts under these conditions. Given the established specificity of CIS-R107K for blocking the GHR signaling inhibitory actions of CIS, but not those of other SOCS/CIS family members, these findings implicate CIS and the proteasome in the control of GHR internalization following receptor activation and suggest that CIS-dependent receptor internalization is a prerequisite for efficient termination of GHR signaling.     

Insulin inhibits growth hormone signaling via the growth hormone receptor/JAK2/STAT5B pathway

Insulin is important for maintaining the responsiveness of the liver to growth hormone (GH). Insulin deficiency results in a decrease in liver GH receptor (GHR) expression, which can be reversed by insulin administration. In osteoblasts, continuous insulin treatment decreases the fraction of cellular GHR localized to the plasma membrane. Thus, it is not clear whether hyperinsulinemia results in an enhancement or inhibition of GH action. We asked whether continuous insulin stimulation, similar to what occurs in hyperinsulinemic states, results in GH resistance. Our present studies suggest that insulin treatment of hepatoma cells results in a time-dependent inhibition of acute GH-induced phosphorylation of STAT5B. Whereas total protein levels of JAK2 were not reduced after insulin pretreatment for 16 h, GH-induced JAK2 phosphorylation was inhibited. There was a concomitant decrease in GH binding and a reduction in immunoreactive GHR levels following pretreatment with insulin for 8-24 h. In summary, continuous insulin treatment in rat H4 hepatoma cells reduces GH binding, immunoreactive GHR, GH-induced phosphorylation of JAK2, and GH-induced tyrosine phosphorylation of STAT5B. These findings suggest that hepatic GH resistance may develop when a patient exhibits chronic hyperinsulinemia, a condition often observed in patients with obesity and in the early stage of Type 2 diabetes.     

Desensitization of the JAK2/STAT5 GH signaling pathway associated with increased CIS protein content in liver of pregnant mice

Chronic exposure to growth hormone (GH) was related to the desensitization of the JAK2/STAT5 signaling pathway in liver, as demonstrated in cells, female rats, and transgenic mice overexpressing GH. The cytokine-induced suppressor (CIS) is considered a major mediator of this desensitization. Pregnancy is accompanied by an increment in GH circulating levels, which were reported to be associated with hepatic GH resistance, although the molecular mechanisms involved in this resistance are not clearly elucidated. We thus evaluated the JAK2/STAT5b signaling pathway and its regulation by the suppressors of cytokine signaling (SOCS)/CIS family and the JAK2-interacting protein SH2-Bbeta in pregnant mouse liver, a model with physiological prolonged exposure to high GH levels. Basal tyrosyl phosphorylation levels of JAK2 and STAT5b in pregnant mice were similar to values obtained for virgin animals, in spite of the important increment of GH they exhibit. Moreover, these signaling mediators were not phosphorylated upon GH stimulation in pregnant mice. A 3.3-fold increase of CIS protein content was found for pregnant mice, whereas the abundance of the other SOCS proteins analyzed and SH2-Bbeta did not significantly change compared with virgin animals. The desensitization of the JAK2/STAT5b GH signaling pathway observed in pregnant mice would then be mainly related to increased CIS levels rather than to the other regulatory proteins examined.     

Interaction of fibroblast growth factor receptor 3 and the adapter protein SH2-B. A role in STAT5 activation

Fibroblast growth factor receptor 3 (FGFR3) influences a diverse array of biological processes, including cell growth, differentiation, and migration. Activating mutations in FGFR3 are associated with multiple myeloma, cervical carcinoma, and bladder cancer. To identify proteins that interact with FGFR3 and which may mediate FGFR3-dependent signaling, a yeast two-hybrid screen was employed using the cytoplasmic kinase domain of FGFR3 as bait. We identified the adapter protein SH2-B as an FGFR3-interacting protein. Coimmunoprecipitation experiments demonstrate binding of the SH2-B beta isoform to FGFR3 in 293T cells. Tyrosine phosphorylation of SH2-B beta was observed when coexpressed with activated FGFR3 mutants such as the weakly activated mutant N540K or the strongly activated mutant K650E, both associated with human developmental syndromes. The extent of tyrosine phosphorylation of SH2-B beta correlates with receptor activation, suggesting that FGFR3 activation mediates tyrosine phosphorylation of SH2-B beta. Furthermore, two tyrosine phosphorylation sites of FGFR3, Tyr-724 and Tyr-760, are required for optimal binding of the Src homology-2 (SH2) domain of SH2-B beta. We also demonstrate the phosphorylation and nuclear translocation of Stat5 by activated FGFR3, which increases in response to overexpression of SH2-B beta. Taken together, our results identify SH2-B beta as a novel FGFR3 binding partner that mediates signal transduction.     

Methylsulfonylmethane suppresses breast cancer growth by down-regulating STAT3 and STAT5b pathways

Breast cancer is the most aggressive form of all cancers, with high incidence and mortality rates. The purpose of the present study was to investigate the molecular mechanism by which methylsulfonylmethane (MSM) inhibits breast cancer growth in mice xenografts. MSM is an organic sulfur-containing natural compound without any toxicity. In this study, we demonstrated that MSM substantially decreased the viability of human breast cancer cells in a dose-dependent manner. MSM also suppressed the phosphorylation of STAT3, STAT5b, expression of IGF-1R, HIF-1α, VEGF, BrK, and p-IGF-1R and inhibited triple-negative receptor expression in receptor-positive cell lines. Moreover, MSM decreased the DNA-binding activities of STAT5b and STAT3, to the target gene promoters in MDA-MB 231 or co-transfected COS-7 cells. We confirmed that MSM significantly decreased the relative luciferase activities indicating crosstalk between STAT5b/IGF-1R, STAT5b/HSP90α, and STAT3/VEGF. To confirm these findings in vivo, xenografts were established in Balb/c athymic nude mice with MDA-MB 231 cells and MSM was administered for 30 days. Concurring to our in vitro analysis, these xenografts showed decreased expression of STAT3, STAT5b, IGF-1R and VEGF. Through in vitro and in vivo analysis, we confirmed that MSM can effectively regulate multiple targets including STAT3/VEGF and STAT5b/IGF-1R. These are the major molecules involved in tumor development, progression, and metastasis. Thus, we strongly recommend the use of MSM as a trial drug for treating all types of breast cancers including triple-negative cancers.     

Suppressor of cytokine signaling 7 inhibits prolactin, growth hormone, and leptin signaling by interacting with STAT5 or STAT3 and attenuating their nuclear translocation

We report here the role of one of the less studied members of the family of suppressors of cytokine signaling (SOCS), namely SOCS-7, in cytokine signaling. We demonstrate that SOCS-7 inhibits prolactin (PRL), growth hormone (GH), or leptin (LEP) signaling mediated through STAT3 and STAT5 in a dose-dependent manner. SOCS-7 also attenuated STAT3 and STAT5 signaling induced by overexpression of JH1, the catalytic subdomain of JAK2. Since SOCS-7 interacted with phosphorylated STAT3 or STAT5, we assumed that SOCS-7 acts at the level of STAT proteins. Indeed, we showed that SOCS-7 inhibits PRL- and leptin-induced STAT5 and STAT3 phosphorylation and prevented the nuclear translocation of activated STAT3. Taken together, our results indicate that SOCS-7 is a physiological dysregulator of PRL, leptin, and probably also GH signaling and that its mode of action is a novel variation of SOCS protein inhibition of cytokine-inducible STAT-mediated signal transduction.     

Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling.

Activation of the tyrosine kinase JAK2 is an essential step in cellular signaling by growth hormone (GH) and multiple other hormones and cytokines. Murine JAK2 has a total of 49 tyrosines which, if phosphorylated, could serve as docking sites for Src homology 2 (SH2) or phosphotyrosine binding domain-containing signaling molecules. Using a yeast two-hybrid screen of a rat adipocyte cDNA library, we identified a splicing variant of the SH2 domain-containing protein SH2-B, designated SH2-Bbeta, as a JAK2-interacting protein. The carboxyl terminus of SH2-Bbeta (SH2-Bbetac), which contains the SH2 domain, specifically interacts with kinase-active, tyrosyl-phosphorylated JAK2 but not kinase-inactive, unphosphorylated JAK2 in the yeast two-hybrid system. In COS cells coexpressing SH2-Bbeta or SH2-Bbetac and murine JAK2, both SH2-Bbetac and SH2-Bbeta coimmunoprecipitate to a significantly greater extent with wild-type, tyrosyl-phosphorylated JAK2 than with kinase-inactive, unphosphorylated JAK2. SH2-Bbetac also binds to immunoprecipitated wild-type but not kinase-inactive JAK2 in a far Western blot. In 3T3-F442A cells, GH stimulates the interaction of SH2-Bbeta with tyrosyl-phosphorylated JAK2 both in vitro, as assessed by binding of JAK2 in cell lysates to glutathione S-transferase (GST)-SH2-Bbetac or GST-SH2-Bbeta fusion proteins, and in vivo, as assessed by coimmunoprecipitation of JAK2 with SH2-Bbeta. GH promoted a transient and dose-dependent tyrosyl phosphorylation of SH2-Bbeta in 3T3-F442A cells, further suggesting the involvement of SH2-Bbeta in GH signaling. Consistent with SH2-Bbeta being a substrate of JAK2, SH2-Bbetac is tyrosyl phosphorylated when coexpressed with wild-type but not kinase-inactive JAK2 in both yeast and COS cells. SH2-Bbeta was also tyrosyl phosphorylated in response to gamma interferon, a cytokine that activates JAK2 and JAK1. These data suggest that GH-induced activation and phosphorylation of JAK2 recruits SH2-Bbeta and its associated signaling molecules into a GHR-JAK2 complex, thereby initiating some as yet unidentified signal transduction pathways. These pathways are likely to be shared by other cytokines that activate JAK2.     

Negative regulation of growth hormone receptor/JAK2 signaling by signal regulatory protein alpha

Signal regulatory proteins (SIRPs) are receptor-like transmembrane proteins, the majority of which contain a cytoplasmic proline-rich region and four cytoplasmic tyrosines that, when phosphorylated, bind SH2 domain-containing protein tyrosine phosphatases (SHP). We demonstrated previously that growth hormone (GH) induces tyrosyl phosphorylation of SIRPalpha and association of SIRPalpha with SHP-2. The GH-activated tyrosine kinase JAK2 associates with and tyrosyl-phosphorylates SIRPalpha1. Here we show that JAK2-SIRPalpha1 association does not require phosphotyrosines in SIRPalpha1 or JAK2 or the proline-rich region of SIRPalpha1. However, when the C-terminal 30 amino acids of SIRPalpha1 containing the proline-rich region and tyrosine 495 are deleted, tyrosyl phosphorylation of SIRPalpha1 by JAK2 and association of SHP-2 with SIRPalpha1 are reduced. GH-dependent tyrosyl phosphorylation of JAK2 is reduced when wild-type SIRPalpha1 compared with SIRPalpha1 lacking the four cytoplasmic tyrosines (SIRP 4YF) is expressed in cells, suggesting that SIRPalpha1 negatively regulates GHR/JAK2 signaling. Consistent with reduced JAK2 activity, overexpression of wild-type SIRPalpha1 but not SIRP 4YF reduces GH-induced phosphorylation of ERKs 1 and 2, STAT3, and STAT5B. These results suggest that SIRPalpha1 is a negative regulator of GH signaling and that the ability of SIRPalpha1 mutants to negatively regulate GHR-JAK2 signaling correlates with their ability to bind SHP-2.     

Hendra virus V protein inhibits interferon signaling by preventing STAT1 and STAT2 nuclear accumulation

The V protein of the recently emerged paramyxovirus, Nipah virus, has been shown to inhibit interferon (IFN) signal transduction through cytoplasmic sequestration of cellular STAT1 and STAT2 in high-molecular-weight complexes. Here we demonstrate that the closely related Hendra virus V protein also inhibits cellular responses to IFN through binding and cytoplasmic sequestration of both STAT1 and STAT2, but not STAT3. These findings demonstrate a V protein-mediated IFN signal evasion mechanism that is a general property of the known Henipavirus species.